1. Field of the Invention
The invention relates to genes which control sporulation in mycobacteria. In particular the invention relates to the whm gene family. The invention also relates to compositions and methods for the prophylaxis, diagnosis and treatment of diseases caused by mycobacteria.
2. Background Information
The unusually slow growth rate of mycobacteria is significant to both the pathogenesis and treatment of mycobacterial infections, such as tuberculosis and leprosy. The slow growth rate necessitates protracted chemotherapeutic regimens which leads to poor patient compliance and the emergence of drug-resistant organisms. Little is known about the mechanism of cell division and its regulation in these bacteria.
Multiple proteins, including the highly conserved tubulin-like protein FtsZ, are known to participate in assembly of the prokaryotic cell division apparatus (1-3). The interactions among these proteins and the temporal and spatial regulation of their functions in Escherichia coli, Bacillus subtilis, and Caulobacter crescentus are the focus of intensive investigation (4). Doubling times for mycobacteria are significantly longer than in these better characterized species: 18-24 hours in Mycobacterium tuberculosis, 14 days in Mycobacterium leprae, and 3 hours in Mycobacterium smegmatis (a saprophyte). The basis for these prolonged cell division times is unknown, although the limited number of rRNA operons (one in M. tuberculosis, two in M. smegmatis) and the metabolic costs of maintaining the complex cell wall that characterizes this genus have been cited as possible contributing factors (5-7).
WhiB is an 87 amino acid protein in Streptomyces coelicolor that is dispensible for normal growth but is required for the maturation of aerial hyphae during sporulation (8,9). S. coelicolor whiB mutants fail to assemble FtsZ rings in their aerial hyphae leading to the arrest of hyphal development prior to crosswall formation (10). The WhiB protein contains an acidic N-terminus with 4 cysteine residues, a helix-turn-helix motif, and a basic C terminus. The mechanism by which WhiB regulates spore formation has not yet been established.
Southern blot surveys suggested the existence of a mycobacterial gene closely related to whiB of S. coelicolor (11). This suggested to the present inventors that such a gene, if present, would be valuable in the diagnosis and treatment of mycobacterial diseases such as tuberculosis.
It is an object of the invention to provide a DNA sequence encoding a protein required for cell division in mycobacterium. Such mycobacteria include, but are not limited to M. tuberculosis, M. bovis and M. smegmatis. The invention provides mycobacterial homologues of the whiB gene from Streptomyces coelicolor. In particular, the invention provides a homologue to the whiB gene, whmD, which is useful for diagnosis and therapy of mycobacterial diseases such as tuberculosis, Mycobacterium avium complex (MAC) infection, leprosy, and infection by atypical mycobacteria.
The whmD gene can be used to screen for drugs which interrupt cell division.
The whmD gene can be crystallized, its 3-dimensional structure determined, and rational drug design employed to develop antimicrobials which are cell division inhibitors for mycobacteria.
The whmD gene can also be used to find useful proteins which interact or bind to it. Among such proteins are likely to be found proteins which are involved in cell division and may also serve as targets for antimicrobial drugs.
The whmD gene may be used as a probe to elucidate cell division in pathogenic mycobacteria. These organisms divide unusually slowly, and may enter a non-replicating state during latent human tuberculosis. Understanding latent tuberculosis is essential for controlling tuberculosis, and should lead to improved vaccines, diagnostics and therapeutic compositions and methods.
The whmD gene may be used to construct a conditionally lethal mutant strain of M. tuberculosis which might be used as a novel live attenuated vaccine against TB. The vaccine strain would be viable in the presence of a chemical inducer which would be co-administered with the vaccine. At an appropriate time after effective immunity had been elicited, the administration of the exogenous chemical inducer would be stopped and the conditionally lethal mutant would die within the host. Such mutants would have advantages over the existing TB vaccine, bacille Calmette-Guerin.
In one embodiment of the invention an isolated and purified subgenomic DNA segment is provided. Its nucleotide sequence is shown in FIG. 2 (SEQ ID NO:1).
In another embodiment of the invention, an isolated and purified amino acid sequences of the whmD protein is provided. Sequences from M. tuberculosis and M. smegmatis are shown in FIG. 3 (SEQ ID NO: 2 and SEQ ID NO:3).
In another embodiment of the invention, a preparation of an isolated polypeptide is provided which comprises at least twelve, preferably at least 20, and most preferably at least 30 contiguous amino acids of the sequence shown in SEQ ID NO:2 or SEQ ID NO:3. Peptides comprising contiguous residues 1-60 of the N-terminal region (MSYES . . . AEEDQ); 61-99 of the central cysteine-rich region (WQER . . . CEVRDAC); 100-126 of the central helix-turn-helix region are considered to be of particular interest.
It is yet another object of the invention to provide a method for screening potential therapeutic agents for the ability to regulate the growth of mycobacteria, particularly M. tuberculosis. 
In yet another embodiment of the invention a reporter construct is provided. The reporter comprises a whmD transcription regulatory region covalently linked in a cis configuration 5xe2x80x2 of a gene encoding an assayable product, wherein transcription of the gene is regulated by the whmD transcription regulatory region.
In another embodiment of the invention a method is provided for screening potential therapeutic agents for the ability inhibit the growth of mycobacterium by inactivating or inhibiting the expression of whmD. The method comprises the steps of: incubating a potential therapeutic agent with a cell which contains a whmD reporter construct, said reporter construct comprising a whmD transcription regulatory region covalently linked in a cis configuration to a downstream gene encoding an assayable product; and measuring the production of the assayable product, a potential therapeutic agent which decreases the production by the cell of the assayable product being an agent which will inhibit the growth of the mycobacterium by inactivating the expression of whmD.
In still another embodiment of the invention a method is provided for screening potential therapeutic agents for use in modulating the growth of a mycobacterium by regulating the activity of whmD. The method comprises the steps of: measuring in vitro transcription from the transcription construct incubated with whmD in the presence or absence of a test compound, the transcription construct comprising a gene coding sequence and a promoter which is responsive to whmD, the promoter being upstream from and adjacent to the gene, the in vitro transcription being effected in the presence and absence of a test substance; and determining whether transcription of the gene is altered by the presence of said test substance, a test substance which alters the transcription of the gene being a candidate for use in regulating the growth of mycobacterium.
In another embodiment of the invention a method is provided for screening potential therapeutic agents for the ability to inhibit the growth of mycobacterium by inhibiting the expression of whmD. The method comprises the steps of: incubating a potential therapeutic agent with a cell which contains a whmD reporter construct, said reporter construct comprising a whmD transcription regulatory region covalently linked in a cis configuration to a downstream gene encoding an assayable product; and measuring the production of the assayable product, a potential therapeutic agent which increases the production by the cell of the assayable product being an agent which will inhibit the growth of M. tuberculosis by inhibiting the expression of whmD.
In still another embodiment of the invention a method is provided for screening potential therapeutic agents for use in modulating the growth of mycobacteria by regulating the activity of whmD. The method comprises the steps of: measuring in vitro transcription from the transcription construct incubated with whmD in the presence or absence of a test compound, the transcription construct comprising a gene coding sequence and a promoter which is responsive to whmD, the promoter being upstream from and adjacent to the gene, the in vitro transcription being effected in the presence and absence of a test substance; and determining whether transcription of the gene is altered by the presence of said test substance, a test substance which alters the transcription of the gene being a candidate for use in regulating the growth of mycobacteria.
In another embodiment of the invention, the whmD protein can be used for rational drug design. The binding targets and protein co-factors of whmD can be determined; the three dimensional structure of whmD can be determined by X-ray crystallography, computer aided design can be used to contruct small-molecule inhibitors of whmD binding to target substrates and cofactors. Since whmD is an essential gene, these inhibitors will be bacteriocidal.
These and other embodiments of the invention provide the art with diagnostic, therapeutic and prophylactic reagents and methods for combating mycobacterial diseases such as tuberculosis, and reagents and methods for identifying therapeutic agents to treat such diseases.
Methods for measuring transcriptional or translational activity in vivo can be any which are known in the art. For example, a primer extension assay may be employed to measure the transcription of the reporter gene. The translation of the reporter gene may be measured by determining the activity of the translation product of the reporter gene. Methods for measuring the activity of an assayable product of certain reporter genes are well known in the art.
Potential therapeutic agents can also be screened for use in regulating the growth of mycobacteria by their ability to regulate the activity of whmD protein. The ability of a test compound or a potential therapeutic agent to regulate the activity of whmD protein is assessed by measuring the transcription of a promoter in an in vitro transcription assay.
A transcription reaction comprises a promoter, responsive to whmD protein and a gene. The gene in the transcription construct could be any gene known in the art. In a preferred embodiment, the length of the promoter region to be tested is less than 200 bp and no more than 600 bp. The promoter in the transcription construct can be any to which whmD protein binds and which it activates or represses. The promoter is responsive to whmD protein which induces the transcription of the gene downstream from and adjacent to the promoter.
Suitable methods for measuring in vitro transcription are any known in the art. In vitro transcription may be carried out by incubating a transcription construct with whmD protein, labeled nucleotides, e.g., 32P-ATP, core RNA polymerase, nucleotides, and buffer reagents in the presence and absence of a test compound (12). The procedures for purifying core RNA polymerase from mycobacteria are well-described in the art (13). The conditions for in vitro transcription are also well known in the art (14). The labeled transcript can be detected by gel electrophoresis and measured by any technique known in the art.
A potential therapeutic agent which decreases the production of the assayable product in the cell indicates its ability to decrease the expression of whmD. Test compounds which decrease the expression of the whmD gene or the activity of the whmD protein can inhibit the growth of mycobacterium by blocking cell division. These compounds can be administered to a human with active mycobacterial infection.
whmD is postulated to be an essential gene in M. tuberculosis and accordingly cannot be knocked out in the true sense. However, it should be possible to knock out whmD in the presence of a conditionally complementing whmD gene wherein a drug-sensitive promoter is used to keep the conditionally complemented strain viable. The strain could be maintained by the drug for as long as desired, and would die when the drug is withdrawn. A host could be immunized by administration of such a mutant strain along with the drug, and once immunization is complete, the drug withdrawn so that the vaccine strain dies. Such mutations can be made by any means known in the art, e.g., PCR, restriction digestion, in vitro or in vivo mutagenesis.
These and aspects of the invention are more fully described below. The examples are provided for exemplification purposes only and are not intended to limit the scope of the invention.